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An improved assay for rapid detection of viable Staphylococcus aureus cells by incorporating surfactant and PMA treatments in qPCR

BACKGROUND: Staphylococcus aureus is an important human pathogen causing a variety of life-threatening diseases. Rapid and accurate detection of Staphylococcus aureus is a necessity for prevention of outbreaks caused by this pathogen. PCR is a useful tool for rapid detection of foodborne pathogens,...

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Autores principales: Zi, Chen, Zeng, Dexin, Ling, Nan, Dai, Jianjun, Xue, Feng, Jiang, Yuan, Li, Baoguang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6182795/
https://www.ncbi.nlm.nih.gov/pubmed/30309323
http://dx.doi.org/10.1186/s12866-018-1273-x
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author Zi, Chen
Zeng, Dexin
Ling, Nan
Dai, Jianjun
Xue, Feng
Jiang, Yuan
Li, Baoguang
author_facet Zi, Chen
Zeng, Dexin
Ling, Nan
Dai, Jianjun
Xue, Feng
Jiang, Yuan
Li, Baoguang
author_sort Zi, Chen
collection PubMed
description BACKGROUND: Staphylococcus aureus is an important human pathogen causing a variety of life-threatening diseases. Rapid and accurate detection of Staphylococcus aureus is a necessity for prevention of outbreaks caused by this pathogen. PCR is a useful tool for rapid detection of foodborne pathogens, however, its inability to differentiate DNA from dead cells and live cells in amplification severely limits its application in pathogen detection. The aim of this study was to develop an improved assay was developed by incorporating the sample treatments with a surfactant and propidium monoazide (PMA) in qPCR for detection of viable S. aureus cells. RESULTS: The cell toxic effect testing with the two surfactants showed that the viability of S. aureus was virtually not affected by the treatment with 0.5% triton x-100 or 0.025% sarkosyl. Triton x-100 was coupled with PMA for sample treatments for detection of viable S. aureus cells in artificially contaminated milk. The qPCR results indicated that the assay reached high an amplification efficiency of 98.44% and the live S. aureus cells were accurately detected from the triton-treated spiked milk samples by the PMA-qPCR assay. CONCLUSIONS: The qPCR assay combined with treatments of PMA and surfactants offers a sensitive and accurate means for detection of viable S. aureus cells. Cell toxic effect testing with the two surfactants showed that the viability of S. aureus was virtually not affected by the treatment with 0.5% triton x-100 or 0.025% sarkosyl. The information on sample treatment with surfactants to improve the dead cell DNA removal efficiency in qPCR by increasing PMA’s permeability to dead cells can be used for other pathogens, especially for Gram-positive bacteria.
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spelling pubmed-61827952018-10-18 An improved assay for rapid detection of viable Staphylococcus aureus cells by incorporating surfactant and PMA treatments in qPCR Zi, Chen Zeng, Dexin Ling, Nan Dai, Jianjun Xue, Feng Jiang, Yuan Li, Baoguang BMC Microbiol Methodology Article BACKGROUND: Staphylococcus aureus is an important human pathogen causing a variety of life-threatening diseases. Rapid and accurate detection of Staphylococcus aureus is a necessity for prevention of outbreaks caused by this pathogen. PCR is a useful tool for rapid detection of foodborne pathogens, however, its inability to differentiate DNA from dead cells and live cells in amplification severely limits its application in pathogen detection. The aim of this study was to develop an improved assay was developed by incorporating the sample treatments with a surfactant and propidium monoazide (PMA) in qPCR for detection of viable S. aureus cells. RESULTS: The cell toxic effect testing with the two surfactants showed that the viability of S. aureus was virtually not affected by the treatment with 0.5% triton x-100 or 0.025% sarkosyl. Triton x-100 was coupled with PMA for sample treatments for detection of viable S. aureus cells in artificially contaminated milk. The qPCR results indicated that the assay reached high an amplification efficiency of 98.44% and the live S. aureus cells were accurately detected from the triton-treated spiked milk samples by the PMA-qPCR assay. CONCLUSIONS: The qPCR assay combined with treatments of PMA and surfactants offers a sensitive and accurate means for detection of viable S. aureus cells. Cell toxic effect testing with the two surfactants showed that the viability of S. aureus was virtually not affected by the treatment with 0.5% triton x-100 or 0.025% sarkosyl. The information on sample treatment with surfactants to improve the dead cell DNA removal efficiency in qPCR by increasing PMA’s permeability to dead cells can be used for other pathogens, especially for Gram-positive bacteria. BioMed Central 2018-10-11 /pmc/articles/PMC6182795/ /pubmed/30309323 http://dx.doi.org/10.1186/s12866-018-1273-x Text en © The Author(s). 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Methodology Article
Zi, Chen
Zeng, Dexin
Ling, Nan
Dai, Jianjun
Xue, Feng
Jiang, Yuan
Li, Baoguang
An improved assay for rapid detection of viable Staphylococcus aureus cells by incorporating surfactant and PMA treatments in qPCR
title An improved assay for rapid detection of viable Staphylococcus aureus cells by incorporating surfactant and PMA treatments in qPCR
title_full An improved assay for rapid detection of viable Staphylococcus aureus cells by incorporating surfactant and PMA treatments in qPCR
title_fullStr An improved assay for rapid detection of viable Staphylococcus aureus cells by incorporating surfactant and PMA treatments in qPCR
title_full_unstemmed An improved assay for rapid detection of viable Staphylococcus aureus cells by incorporating surfactant and PMA treatments in qPCR
title_short An improved assay for rapid detection of viable Staphylococcus aureus cells by incorporating surfactant and PMA treatments in qPCR
title_sort improved assay for rapid detection of viable staphylococcus aureus cells by incorporating surfactant and pma treatments in qpcr
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6182795/
https://www.ncbi.nlm.nih.gov/pubmed/30309323
http://dx.doi.org/10.1186/s12866-018-1273-x
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